Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTSDisrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1b release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1b, interferon-g and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONSColchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action -inhibition of pore formation induced by activation of P2X receptors -which could explain some of the anti-inflammatory effects of colchicine. LINKED ARTICLEThis article is commented on by Pelegrín, pp. 908-911 of this issue. To view this commentary visit http://dx.doi.org/10.1111/ j. 1476-5381.2011.01325.x Abbreviations DAPI, 4′,6-diamidino-2-phenylindole; DCFH2DA, dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; EB, ethidium bromide; IFN-g, interferon-g; IL-1b, interleukin-1b; LPS, lipopolysaccharide; PGP, P-glycoprotein; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species BJP British Journal of Pharmacology

ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B4 (LTB4) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB4 is produced upon P2X7 receptor activation and examined whether LTB4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)–deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB4, and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB4 formation, which is required for L. amazonensis elimination.

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.

SummaryNucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including antiparasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca 2+ changes. Infection of macrophages with Leishmania upregulated the expression of P2Y2 and P2Y4 receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.